1. Field of the Invention
This invention relates to the field of hematology, particularly to differentiating subclasses of white blood cells.
2. Brief Description of the Prior Art
Human white blood cells are classified as lymphocytes, monocytes and polymorphonuclear cells (PMNs)o PMNs are sub-classified as neutrophils, eosinophils or basophils based on the staining characteristics of their cytoplasmic granules.
Differentiation of white blood cells has commonly been accomplished by various staining techniques. Several phthalocyanin compounds are known for use as such. The first of these to be discovered is called Alcian Blue. Alcian Blue has been used for the differential staining of basophils. The copper phthalocyanin cationic dyes are not sufficiently specific to achieve the selective staining of basophils when used alone because they also stain other cells which possess polynucleotides, e.g., DNA and RNA. In addition, basophils stain because of the unique presence in them of heparin, a sulfated polysaccharide. One way of establishing the desired selectivity is to combine it with lanthanum chloride which masks the polynucleotide phosphate groups and thereby prevents them from binding the phthalocyanin anion.
The use of Alcian Blue requires a closely controlled, highly acidic pH and it is heat labile. At alkaline pH and when exposed to heat, Alcian Blue forms particulates (insoluble dyes). This tendency to precipitate has been a longstanding problem in Alcian Blue-containing reagents. Automated analysis instruments contain components such as filters which collect these precipitates. This can interfere with the reliability of the determinations being made and even the operation of instruments on which this method is performed. It has nonetheless been considered the dye of choice because of its specificity and distinct color. For more background information on Alcian Blue, see Gilbert, et al, Basophil Counting With A New Staining Method Using Alcian Blue, Blood, 46:279-286 (1975).
Other phthalocyanin dyes have since been developed. For example, Bloom, et al, Histochemie, 2:48-57 (1960) shows the use of underivatized Astra Blue (free base) to stain biological tissues containing mucopolysaccharides, particularly mast cells. The Astra Blue free base is used in 0.5N HCl which gives it a positive charge. The low pH allows selectivity because of the inherent strength of sulfuric acid derivatives, e.g., heparin, which is ionized at pH 0.3, as compared to the weakness of phosphoric acid derivatives, e.g., DNA, which is not ionized at low pH.
Inagaki, Acta Hematologica Japonica, 32 (4):642-647 (1969), describes a method for staining basophil and mast cell granules using free base Astra Blue and a fixative solution of Acridine Orange in methanol containing 0.5M NaCl. Inagaki examined saturated cetyl pyridinium chloride in absolute methanol and saturated Acridine in absolute methanol for the fixation of peripheral blood and bone marrow smears. Cetyl pyridinium chloride securely preserved the basophil granules and the mast cell granules, but the Astra Blue staining tended to be prevented. Acridine could not preserve these cell granules sufficiently in the above described procedure.
In summary, Alcian Blue and Astra Blue free base and its quaternary derivatives have been the only compounds of this type which have been known to differentiate basophils from other white blood cells. The instability of Alcian Blue reagent has been a longstanding problem. Thus, workers in the field have continued to search for compounds which selectively stain basophils, in contrast to other white blood cells. Further, dye uptake is dependent on each individual user's staining technique.
Since cellular maturation is a continuous process, the successive stages involved are difficult to differentiate. However, separate stages can be recognized in whole blood smears stained with Wright's or Giemsa stains. This classification is based on the presence, nature and number of granules and the cytoplasmic and nuclear characteristics of each cell. These classifications of white blood cells and techniques for their differentiation are well known. See, for example, Ansley, et al, U.S. Pat. No. 3,741,875. However, classification of stained, intact cells based on cytoplasmic and nuclear information is very dependent upon subjective characterization by the user.
Kim, U.S. Pat. No. 4,099,917, has disclosed a method of preparing a blood sample for discrimination between classes of unstained white blood cells by their cell size and granularity characteristics. A blood sample is treated with a detergent which lyses red blood cells but does not lyse white cells, a fixative is added and the preparation is incubated. The cell suspension so obtained is said to allow differentiation of unstained, fixed, intact white cells by optical systems having low and high angle light scatter characteristics. This requires large complex instrumentation and, thus cannot be done by visual observation.
Ledis, et al, U.S. Pat. No. 4,286,963 discloses a composition comprised of (a) at least one long chain alkyl trimethyl quaternary ammonium salt, such as hexadecyl trimethyl ammonium bromide and (b) at least one additive selected from (i) a short chain alkanol substituted by phenyl or phenoxy, such as 2-phenoxyethanol and (ii) a polyhydroxy compound such as sorbitol for lysing red cells so that a differential determination of lymphoid and myeloid populations of white blood cells can be made.
Thus, most of the known techniques for this differentiation require the preparation and use of stains or provide for lysis only of red blood cells. Some require complex instrumentation. Otherwise, reported differentiation in whole cells, whether stained or unstained, is very much dependent upon subjective characterization. Nothing in the literature describes a reliable method for simultaneously determining an accurate basophil count and a lobularity index in the same sample of treated blood and in the absence of a stain.
Groner, et al, Blood Cells, 6:141-157 (1980) discusses differentiation of white blood cell subclasses using optical scatter, staining properties and other techniques. The concept of "left shift" is mentioned, referring to a trend in neutrophil populations toward more immature or less lobulated forms. A sharp change in index of refraction was created at the nuclear boundary by treating a whole blood sample with a strong cationic detergent and maleic acid. As a result, the red blood cells were lysed, most of the cytoplasm of the leukocytes was leached, and the nucleus shrank slightly. From the discussion in this reference, it appears that the leukocyte membranes were not ruptured or lysed (as mentioned with reference to red blood cells), leukocyte cytoplasm was not completely (only mostly) stripped leaving artifacts which distort the apparent shape of the nucleus and, finally, there is no mention of differentiation of the effect of this treatment between one leukocyte subclass and any other.